Biology subjects

Brown, R. J. P.

Publications and source records attributed to Brown, R. J. P..

7 recordsLinked to original sources

bioRxiv2026

Yellow fever vaccine propagation in primary human hepatocytes triggers antiviral and cytolytic responses

Yellow fever virus (YFV) infection can cause severe-to-fatal liver damage in humans, while immunization with the attenuated 17D vaccine strain has an excellent safety record, priming protective host immunity in the absence of pathology. To investigate virus-host correlates associated with these differential clinical outcomes, we combined YFV genome-level evolutionary analyses with investigations of vaccine and virulent strain hepatotropism. Evolutionary analyses confirmed purifying selection is the dominant force driving global YFV genome divergence, with functional constraints associated with the YFV transmission cycle selecting against vaccine attenuating mutations in virulent strains. In immune deficient hepatoma cells, 17D exhibited enhanced early propagation, spreading and apoptosis induction when compared to virulent strains. Ex vivo infections performed in primary human hepatocytes (PHH) from multiple donors confirmed robust propagation of both 17D and virulent YFV strains. RNA-sequencing revealed consistent and shared induction of IFNB and IFNL1-4, modulating overlapping gene programs associated with antiviral responses, immunity, chemotaxis and inflammation, cell-death, metabolic reprogramming and protein translation. Subtle differences in virion production kinetics and the magnitude and tempo of PHH transcriptional responses were observed. Antiviral responses to 17D were activated earlier while responses to virulent YFV were delayed but enhanced, mirroring virus propagation kinetics. More broadly, cellular responses to YFV infection are likely dominated by paracrine IFN signalling, with enriched LRP1 expression coupled with impaired IFN production in PHH contributing to YFVs robust hepatotropism. In summary, we demonstrate comparable propagation kinetics and PHH transcriptional responses to vaccine and virulent YFV strains, highlighting impaired hepatotropism is not a correlate of vaccine attenuation. These data imply that unknown barriers restrict liver access and associated organ pathology upon vaccination with 17D.

microbiology↗

bioRxiv2023

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence. Author SummaryHCV infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms which underly persistence are incompletely defined. We utilized a long-term cell culture adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

molecular biology↗

bioRxiv2023

Targeting cellular cathepsins inhibits hepatitis E virus infection

Background and AimsThe hepatitis E virus (HEV) is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other hepatotropic viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as a class of host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potentials of cellular proteases during HEV infection. Approach and ResultsUsing our recently established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors, impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 robustly suppressed HEV infections with an EC50 of [~] 0.01 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells until micromolar concentrations, was also observed in differentiated HepaRG and ex vivo in primary human hepatocytes. Furthermore, through time-of-addition experiments, we confirmed that HEV entry is potently blocked by inhibition of cathepsins and cathepsin L (CTSL) knockout cells were less permissive to HEV suggesting that CTSL is critical for HEV infection. ConclusionsIn summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor, K11777, especially with its notable safety profile in primary cells, further underscores its potential as a promising therapeutic candidate.

cell biology↗

bioRxiv2023

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs

Factors of the innate immune response to SARS-CoV-2 in the lungs are pivotal for the ability of the host to deal with the infection. In humans, excessive macrophage infiltration is associated with disease severity. Using 3D spatiotemporal analysis of optically cleared hamster lung slices in combination with virological, immunohistochemical and RNA sequence analyses, we visualized the spread of SARS-CoV-2 through the lungs and the rapid anti-viral response in infected lung epithelial cells, followed by a wave of monocyte-derived macrophage (MDM) infiltration and virus elimination from the tissue. These SARS-CoV-2 induced innate immune processes are closely related to the onset of necrotizing inflammatory and consecutive remodelling responses in the lungs, which manifests as extensive cell death, vascular damage, thrombosis, and cell proliferation. Here we show that MDM are directly linked to virus clearance, and appear in connection with tissue injury and blood vessel damage. Rapid initiation of prothrombotic factor upregulation, tissue repair and alveolar cell proliferation results in tissue remodelling, which is followed by fibrosis development despite a decrease in inflammatory and anti-viral activities. Thus, although the hamsters are able to resolve the infection following the MDM influx and repair lung tissue integrity, longer-term alterations of the lung tissues arise as a result of concurrent tissue damage and regeneration processes.

immunology↗

bioRxiv2022

SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

microbiology↗

bioRxiv2021

In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines. We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H). Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.

immunology↗

bioRxiv2021

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of [~]90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity. IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.

immunology↗